New treatment strategies for respiratory diseases: Ex vivo and in vivo evaluation of pharmacological immunomodulations using a nanoparticle-based drug delivery system

Every year, infections with influenza viruses are responsible for high rates of morbidity and mortality, not only but mainly in the high-risk groups. In particular constant mutations within the virus RNA and the frequent occurrence of zoonosis make the virus highly unpredictable and cause limited efficacy of the currently available influenza vaccines. Annual vaccination, however, is the main prevention strategy against this serious worldwide health threat. Yet, the currently available influenza vaccines face some limitations, especially in the case of unforeseen virus subtypes. These limitations include time-consuming production processes, which impede a fast adaptation to new virus subtypes and complicate the production of sufficient vaccine quantities in an adequate time frame to meet the global demand. Thus, improved vaccine strategies are needed which enable adjustment of vaccines to new subtypes and their rapid production. These new vaccines should provide maximal protection against influenza infections, while requiring a minimal dose. Ideally, these vaccines should further meet with high public acceptance to improve global influenza vaccine coverage. The aim of the presented work was the development and characterization of a new inhalable nanoparticle-based influenza vaccine regarding its local toxicity and efficacy. The tested vaccine consists of a recombinant H1N1 influenza hemagglutinin protein (HAC1) which was produced in fast-growing Nicotiana benthamiana plants, enabling fast adjustment to new circulating virus subtypes and production of large quantities. Furthermore, the protein was formulated with silica-nanoparticles (NP) serving as drug delivery system to administer the vaccine directly into the lungs and to induce local protection at the site of virus entry and settlement. For assessment of the local toxicological data of this inhalable influenza vaccine and its property to locally induce an antigen-specific recall response, a reproducible human ex vivo tissue model of precision-cut lung slices (PCLS) was used. This organotypic tissue model reflects the functional heterogeneity of the human lung, the target of this inhalable vaccine. In this tissue model no local cellular toxicity of the vaccine was observed within applicable concentrations. The silica-NP though provoked a dose-dependent induction of pro-inflammatory mediators such as TNF-? and IL-1?, indicating their adjuvant properties. Moreover, the protein induced re-activation of a T cell response, marked by increased levels of IL-2 and IFN-?. The combination of the recombinant protein and the silica-NP in the vaccine induced boosted IFN-? secretion compared with the protein effect alone. The potential of the vaccine to affect the innate and adaptive immune responses within a nontoxic range was further investigated in a murine vaccination model. The focus of this in vivo investigation was on the evaluation of the systemic and local immunogenicity of the vaccine. Additionally to the vaccine tested ex vivo, a second mucosal adjuvant candidate, bis-(3',5')- cyclic dimeric guanosine monophosphate (c-di-GMP), was tested to compare singleadjuvanted vaccines with the effectiveness of a double-adjuvanted vaccine administered at the site of virus infection, the respiratory tract. The systemic antibody response of the locally administered double-adjuvanted vaccine, marked by HA inhibition and HA-specific IgG titers in the serum, was comparable to the systemic vaccination control and exceeded the titers of the single-adjuvanted vaccines. Notably, low doses of the double-adjuvanted vaccine induced local IgA titers measureable in the bronchoalveolar lavage fluid, which was not observed for the single-adjuvanted vaccines or the systemic vaccine control. Mucosal IgA has been shown to be essential for virus neutralization upon secondary virus encounter and crossprotection against drifted virus strains. Additionally and in line with the ex vivo results, the local vaccination with a mucosal adjuvant induced an antigen-specific re-activatable T cell response in PCLS of vaccinated mice. These findings demonstrate the capability of a plant-produced recombinant protein formulated with an adjuvantive drug delivery system to re-activate and boost an adaptive immune response. This potential was even increased by addition of a secondary mucosal adjuvant, leading to mucosal IgG and IgA, a putative line of protection against virus infection in vivo.